GALT

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Introduction

Galactose (Gal) is an essential monosaccharide in the human body, with the Leloir pathway being its principal metabolic route. Four enzymes, galactose mutarotase (GALM), galactokinase 1, (GALK1), galactose 1-phosphate uridylyltransferase (GALT) and UDP-galactose 4′-epimerase (GALE) make up the Leloir pathway, which are responsible for converting galactose from dietary and endogenous sources to glucose for glycolysis, but also interconverts uridine diphosphate (UDP) hexoses for glycogen and glycoconjugate formation. In addition, GALT and GALE are involved in the metabolism of UDP-N-acetyl-hexose-amines, which are substrates for glycosyltransferases and are important structural elements of glycosaminoglycans.

GALT (EC 2.7.7.12) is responsible for the conversion of 1-phosphate galactose (Gal-1-P) and UDP glucose (UDP-Glc) to 1-phosphate glucose (Glc-1-P) and UDP galactose (UDP-Gal). Inherited mutations on the GALT gene cause autosomal recessive classic galactosemia (or type I galactosemia). Newborns with classic galactosemia often present jaundice, cataracts, hepatomegaly, and even death. The only effective treatment is a strict dietary restriction of galactose, but even with diet control, older sufferers develop neurocognitive disabilities in learning and speech, and ovarian failure is common in women with galactosemia.  Hallmarks of classic galactosemia are elevated levels of the metabolite galactose-1-phosphate, reduced levels of UDP-hexose, and disturbed glycosylation, which are thought to contribute to the disease. Currently, 336 mutations are reported in the GALT database, of which ∼60% represent missense changes.

The crystal structure of human GALT (hGALT) has not yet been reported, but the E. coli structure (eGALT) shares 55% sequence identity with the human protein. GALT is a β-chain rich dimeric protein belonging to the histidine triad superfamily. It is a metallo-protein and the eGALT structure shows zinc and iron binding at two different sites. Cys52, Cys55, His115, and His164 are zinc binding sites that play a role in stabilizing the active site. The iron binding sites Glu182, His281, His299, His301 are thought to stabilize the protein structure.

Figure 1. Crystal structure of uridylylated hGALT at 1.9 Å resolution (McCorvie, T.J.; et al. 2016)

Structural basis of classic galactosemia variants

As of January 2016, 178 of the 336 mutations annotated in the GALT database are missense. Overall, variants that may destabilise and misfold the hGALT protein are the most prevalent. They include amino acid changes expected to alter protein flexibility (i.e. p.Pro36Leu, p.Gly83Val/Arg, p.Pro166Ala, p.Leu217Pro), resulting in steric clashes (i.e. p.Cys130Tyr, p.Val168Leu, p.Ala276Asn, p.Leu289Phe/Arg), create cavities (i.e. p.Val125Ala, p.Leu226Val, p.Trp154Gly, p.Met336Leu) and remove hydrogen bonds(i.e. p.Arg67Cys, p.Glu220Lys, p.Gln317Arg/His, p.Arg328His). The second most representative group of variants alters dimer interactions such as interchain salt-bridges. Variants that alter substrate binding are the third most represented, followed by variants that alter metal binding. In addition, some variants located on the surface of the protein have no apparent effect on the protein structure and are called polymorphisms.

The hGALT structure also gave us a detailed molecular view of three variants, p.Ser135Leu, p.Gln188Arg, and p.Lys285Asn, that account for the majority of classical galactosemia. The p.Lys285Asn variant, commonly found in Europeans, removes three hydrogen bonds involving helix α₆, possibly destabilizing the protein as a result. The p.Ser135Leu variant, found only in African ethnicities, removes the interaction with Cys72 and Arg67 and introduced a more hydrophobic and bulkier residue near the uridylation site His186. The most common galactosemia variant (about 60% of patients) p.Gln188Arg affects the active site residue Gln188, which interacts with the covalently linked phosphate and ribose moieties of UMP and the phosphate moiety of hexose-1-phosphate effect. These findings are consistent with previous homology models that predicted altered hydrogen-bonding patterns with the substrate, explaining its low activity.

Figure 2. Structural basis of the most common hGALT variants (McCorvie, T.J.; et al. 2016)